Human Pluripotent Stem Cell-derived Organoids Yield Microvascular-like Endothelial Cells

Introduction: Coronary microvascular disease (CMD) can manifest as microvascular angina or HFpEF and is implicated in post-STEMI remodelling and COVID-19. Superior in vitro models would aid the development of therapies for CMD. This study outlines the creation of a 3D differentiation protocol that yields stable human pluripotent stem cell-derived microvascular-like endothelial cells (hPSC-MVECs). Hypothesis: We hypothesised that the 3D organoid environment would allow for recapitulation of the microvasculature. Methods: Growth factor-mediated endothelial differentiation of hPSCs was conducted for 12 days in stirred tank bioreactors devoid of exogenous matrix substrates such as Matrigel. CD31 pos 3D-derived hPSC-ECs were isolated via fluorescence-activated cell sorting and evaluated via RT-qPCR for their gene expression of endothelial markers relative to 2D-derived hPSC-ECs. 3D hPSC-ECs were subsequently treated with high concentration VEGFA for 7 days (3D+V) and assessed for CD31 expression via high content image analysis. A proteome profiler human angiogenesis array was employed to investigate the angiogenic landscape of the generated hPSC-ECs relative to primary Human Cardiac Microvascular Endothelial Cells (HMVEC-Cs). Transcriptomic analysis of the ECs was conducted via Single-Cell RNA sequencing (scRNA-seq). Results: Organoids attained on day 12 of the 3D differentiation protocol displayed a complex vascular network. Isolated 3D hPSC-ECs displayed comparable levels of PECAM1 and CDH5 relative to 2D hPSC-ECs however, the expression of VEGFA was significantly reduced (P<0.01). The resultant high-concentration treatment with exogenous VEGFA gave rise to phenotypically stable hPSC-ECs that had an angiogenic profile comparable to HMVEC-Cs. scRNA-seq for 3D+V hPSC-ECs revealed expression of several reported microvascular markers including GJA4 (arteriole), S100A4 (post-arteriole capillary), TCF15 (capillary), SOX17 (pre-venular capillary), CD34 (venule), and the coronary vessel marker FABP4 . Conclusions: These data suggest that 3D organoid differentiation facilitates the generation of cardiac microvascular-like ECs herein termed hPSC-MVECs.